RESUMEN
Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4. In this study, we have investigated the binding of such variants and of wild type (wt) cyt C to synthetic cardiolipin-containing vesicles. The mutants have a lower propensity in membrane binding, displaying higher dissociation constants with respect to the wt protein. Compressibility measurements reveal that both variants are more flexible than the wt, suggesting that the native central Ω-loop is important for the interaction with membranes. Such hypothesis is supported by molecular dynamics simulations. A minimal distance analysis indicates that in the presence of cardiolipin the central Ω-loop of the mutants is no more in contact with the membrane, as it happens instead in the case of wt cyt C. Such finding might provide a hint for the reduced membrane binding capacity of the variants and their enhanced peroxidase activity in vivo.
Asunto(s)
Cardiolipinas , Citocromos c , Simulación de Dinámica Molecular , Unión Proteica , Citocromos c/metabolismo , Citocromos c/química , Citocromos c/genética , Humanos , Cardiolipinas/metabolismo , Cardiolipinas/química , Mutación , Membrana Celular/metabolismoRESUMEN
Lipoxygenases (LOXs) are a family of enzymes that includes different fatty acid oxygenases with a common tridimensional structure. The main functions of LOXs are the production of signaling compounds and the structural modifications of biological membranes. These features of LOXs, their widespread presence in all living organisms, and their involvement in human diseases have attracted the attention of the scientific community over the last decades, leading to several studies mainly focused on understanding their catalytic mechanism and designing effective inhibitors. The aim of this review is to discuss the state-of-the-art of a different, much less explored aspect of LOXs, that is, their interaction with lipid bilayers. To this end, the general architecture of six relevant LOXs (namely human 5-, 12-, and 15-LOX, rabbit 12/15-LOX, coral 8-LOX, and soybean 15-LOX), with different specificity towards the fatty acid substrates, is analyzed through the available crystallographic models. Then, their putative interface with a model membrane is examined in the frame of the conformational flexibility of LOXs, that is due to their peculiar tertiary structure. Finally, the possible future developments that emerge from the available data are discussed.
Asunto(s)
Membrana Dobles de Lípidos , Lipooxigenasas , Animales , Humanos , Conejos , Conformación Molecular , Ácidos GrasosRESUMEN
Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).
Asunto(s)
Simulación de Dinámica Molecular , Receptores del Factor de Necrosis Tumoral , Factor 2 Asociado a Receptor de TNF/química , Factor 2 Asociado a Receptor de TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas , FN-kappa B/metabolismo , Unión ProteicaRESUMEN
TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.
Asunto(s)
Subunidades de Proteína/química , Factor 2 Asociado a Receptor de TNF/química , Tirosina/química , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proproteína Convertasas/química , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteína de Dominio de Muerte Asociada a Receptor de TNF/química , Proteína de Dominio de Muerte Asociada a Receptor de TNF/genética , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Termodinámica , Tirosina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and -bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.
Asunto(s)
Aromatasa/genética , Polimorfismo Genético , Espectrometría de Fluorescencia , Aromatasa/metabolismo , Catálisis , Dominio Catalítico , Humanos , Unión ProteicaRESUMEN
The interaction of cytochrome c (cyt c) with natural and synthetic membranes is known to be a complex phenomenon, involving both protein and lipid conformational changes. In this paper, we combined infrared and fluorescence spectroscopy to study the structural transformation occurring to the lipid network of cardiolipin-containing large unilamellar vesicles (LUVs). The data, collected at increasing protein/lipid ratio, demonstrate the existence of a multi-phase process, which is characterized by: (i) the interaction of cyt c with the lipid polar heads; (ii) the lipid anchorage of the protein on the membrane surface; and (iii) a long-distance order/disorder transition of the cardiolipin acyl chains. Such effects have been quantitatively interpreted introducing specific order parameters and discussed in the frame of the models on cyt c activity reported in literature.
Asunto(s)
Cardiolipinas/metabolismo , Citocromos c/metabolismo , Animales , Membrana Celular/metabolismo , Caballos , Espectrometría de Fluorescencia , Espectrofotometría Infrarroja , Liposomas Unilamelares/metabolismoRESUMEN
The oligomeric state of TRAF2 (tumor necrosis factor-receptor associated factor 2), a TNF (tumor necrosis factor) receptor-associated factor, is crucial for membrane binding and probably plays a fundamental role in regulating the protein function in vivo. In this study we have combined molecular dynamics with the protein contact network approach to characterize the interaction of the three identical subunits of TRAF2. The average structure obtained after a 225 ns simulation reveals that two clusters of different size are formed, one of which includes almost completely two subunits, while the third monomer appears to be more independent. This picture is also confirmed by the estimated average number of inter-subunit contacts and by the comparison of side chains mobility in each monomer. The analysis of equilibrium pressure-induced dissociation measurements supports such findings, indicating that the dimeric-monomeric (2 + 1) might be prevalent with respect to the trimeric configuration, especially in the case of more diluted samples. These findings suggest that the formation of monomeric species, which is crucial for the formation of intra-luminal vesicles, might depend on preferential asymmetric interactions among the three subunits.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Simulación de Dinámica Molecular , Receptores del Factor de Necrosis Tumoral , Sustancias Macromoleculares , FN-kappa B/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factores de Necrosis TumoralRESUMEN
The discovery of hemoglobin allosteric properties is briefly summarized and contextualized in the frame of the main biochemical revelations that characterized the first half of the XX century. In particular, the historical background of DNA, RNA, and protein structure research is recalled and the new role that protein-protein interaction may have on allosteric regulation is discussed.
Asunto(s)
Hemoglobinas/química , Hemoglobinas/metabolismo , Regulación Alostérica , Sitio Alostérico , Animales , Humanos , Modelos Moleculares , Conformación Proteica , Mapas de Interacción de ProteínasRESUMEN
Proteins are located in the twilight zone between chemistry and biology, where a peculiar kind of complexity starts. Proteins are the smallest 'devices' showing a sensible adaptation to their environment by the production of appropriate behavior when facing a specific stimulus. This fact qualifies (from the 'effector' side) proteins as nanomachines working as catalysts, motors, or switches. However (from the sensor side), the need to single out the 'specific stimulus' out of thermal noise qualifies proteins as information processing devices. Allostery corresponds to the modification of the configuration (in a broad sense) of the protein molecule in response to a specific stimulus in a non-strictly local way, thereby connecting the sensor and effector sides of the nanomachine. This is why the 'disclosing' of allostery phenomenon is at the very heart of protein function; in this chapter, we will demonstrate how a network-based representation of protein structure in terms of nodes (aminoacid residues) and edges (effective contacts between residues) is the natural language for getting rid of allosteric phenomena and, more in general, of protein structure/function relationships.
Asunto(s)
Proteínas/química , Proteínas/metabolismo , Regulación Alostérica , Bases de Datos de Proteínas , Modelos Moleculares , Conformación Proteica , Mapas de Interacción de Proteínas , Programas InformáticosRESUMEN
In this paper we report a procedure to analyze protein homodimer interfaces.We approached the problem by means of a topological methodology. In particular, we analyzed the subunits interface of about 50 homodimers and we have defined a few parameters that allow to organize these proteins in six different classes. The main characteristics of each class of homodimers have been discussed also taking into account their stabilization energy, as reported in the literature from the experimental measurements. A paradigmatic example for each class has been reported and a graphical representation proposed in order to better explain the meaning of the parameters chosen.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Biología Computacional , Cristalografía por Rayos X , Bases de Datos de Proteínas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
His596 of human ALOX12 has been suggested to interact with the COO--group of arachidonic acid during ALOX catalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange might contribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 for ALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigated the impact of these mutations on structural, catalytic and allosteric enzyme properties. To shed light on the molecular basis of the observed functional alterations we performed in silico substrate docking studies and molecular dynamics simulations and also explored the impact of Gln596 exchange on the protein structure. The combined theoretical and experimental data suggest that Gln596 may not directly interact with the COO--group of arachidonic acid. In contrast, mutations at Gln596 destabilize the secondary and tertiary structure of ALOX15 orthologs, which may be related to a disturbance of the electrostatic interaction network with other amino acids in the immediate surrounding. Moreover, our MD-simulations suggest that the geometry of the dimer interface depends on the structure of substrate bound inside the substrate-binding pocket and that Gln596Ala exchange impairs the allosteric properties of the enzyme. Taken together, these data indicate the structural and functional importance of Gln596 for ALOX15 catalysis.
Asunto(s)
Sitio Alostérico , Araquidonato 15-Lipooxigenasa/química , Simulación del Acoplamiento Molecular , Sustitución de Aminoácidos , Animales , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Ácido Araquidónico/química , Ácido Araquidónico/metabolismo , Estabilidad de Enzimas , Glutamina/química , Glutamina/genética , Humanos , Unión Proteica , Conformación Proteica en Hélice alfa , Conejos , Especificidad por SustratoRESUMEN
The ability of a C-terminal truncated form of TRAF2 to bind synthetic vesicles has been quantitatively studied by steady-state fluorescence energy transfer from the protein to large unilamellar vesicles (LUVs) prepared with different lipid mixtures. The dissociation constants, the free energy of binding, and the average number of phospholipids interacting with truncated TRAF2 have been evaluated from the corresponding binding curves. The results indicate that the protein strongly interacts with the lipid bilayer, preferentially in the monomeric state. These findings have been discussed in terms of their possible role in the activity of TRAF2 in vivo.
Asunto(s)
Membrana Dobles de Lípidos/química , Factor 2 Asociado a Receptor de TNF/química , Sitios de Unión , Transferencia Resonante de Energía de Fluorescencia , Modelos MolecularesRESUMEN
In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state. Generalized Polarization measurements and spectral phasor analysis revealed that truncated-TRAF2 affects the membrane fluidity, especially when vesicles are heated up at physiological temperature. The addition of nanomolar concentration of TRAF2 in GUVs also seems to exert a mechanical action, as demonstrated by the formation of intraluminal vesicles, a process in which ganglioside GM1 plays a crucial role.
Asunto(s)
Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos/química , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Factor 2 Asociado a Receptor de TNF/química , Factor 2 Asociado a Receptor de TNF/metabolismo , Difusión , Fluorescencia , Gangliósido G(M1)/metabolismo , Humanos , Fluidez de la Membrana/fisiología , Unión Proteica/fisiología , Dominios Proteicos , Liposomas Unilamelares/metabolismoRESUMEN
Protein homodimers pose some intriguing questions about the relation between structure and stability. We approached the problem by means of a topological methodology based on protein contact networks. We correlated local interface descriptors with structure and energy global properties of the systems under analysis. We demonstrated that the graph energy, formerly applied to the analysis of unconjugated hydrocarbons structures, is the bridge between the topological and energetic description of protein complexes. This is a first step for the generation of a "protein structural formula", analogous to the molecular graphs in organic chemistry.
Asunto(s)
Conformación Proteica , Multimerización de Proteína , Proteínas/química , Algoritmos , Bases de Datos de Proteínas , Modelos Moleculares , Modelos Estadísticos , Unión Proteica , Mapas de Interacción de Proteínas , Estabilidad Proteica , Subunidades de Proteína , TermodinámicaRESUMEN
TNF receptor-associated factors (TRAFs) are characterized by an oligomeric structure that plays a fundamental role in the binding process with membrane receptors. In this work, we studied the trimer-to-monomer (T â 3M) equilibrium transition of the TRAF2 C-terminal domain using both chemical (dilution/guanidinium hydrochloride) and mechanical stress (high pressure) to induce the dissociation of the native protein into subunits. The experimental results and computer simulations indicate that stable monomers exist and that their population accounts for 15% of the total TRAF2 molecules already at a physiological intracellular concentration (≈1 µM), being instead the predominant species in the nanomolar concentration range. Because the total amount of TRAF2 changes during a cell cycle, the monomer-trimer equilibrium can be crucial for regulating the activities of TRAF2 in vivo.
Asunto(s)
Multimerización de Proteína , Factor 2 Asociado a Receptor de TNF/química , Línea Celular , Humanos , Simulación de Dinámica Molecular , Estabilidad Proteica , Factor 2 Asociado a Receptor de TNF/análisisRESUMEN
The identification of modules in protein structures has major relevance in structural biology, with consequences in protein stability and functional classification, adding new perspectives in drug design. In this work, we present the comparison between a topological (spectral clustering) and a geometrical (k-means) approach to module identification, in the frame of a multiscale analysis of the protein architecture principles. The global consistency of an adjacency matrix based technique (spectral clustering) and a method based on full rank geometrical information (k-means) give a proof-of-concept of the relevance of protein contact networks in structure determination. The peculiar "small-world" character of protein contact graphs is established as well, pointing to average shortest path as a mesoscopic crucial variable to maximize the efficiency of within-molecule signal transmission. The specific nature of protein architecture indicates topological approach as the most proper one to highlight protein functional domains, and two new representations linking sequence and topological role of aminoacids are demonstrated to be of use for protein structural analysis. Here we present a case study regarding azurin, a small copper protein implied in the Pseudomonas aeruginosa respiratory chain. Its pocket molecular shape and its electron transfer function have challenged the method, highlighting its potentiality to catch jointly the structure and function features of protein structures through their decomposition into modules.
Asunto(s)
Modelos Moleculares , Proteínas/química , Azurina/química , Azurina/metabolismo , Biología Computacional , Simulación por Computador , Bases de Datos de Proteínas , Transporte de Electrón , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas/estadística & datos numéricos , Pseudomonas aeruginosa/metabolismoRESUMEN
Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme-membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors.
Asunto(s)
Ácido 5,8,11,14-Eicosatetrainoico/química , Membrana Dobles de Lípidos/química , Inhibidores de la Lipooxigenasa/química , Lipooxigenasa/química , Membranas Mitocondriales/química , Dominio Catalítico , Humanos , Membranas Mitocondriales/enzimologíaRESUMEN
Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for ß-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.
Asunto(s)
Aromatasa/metabolismo , Aromatasa/química , Medición de Intercambio de Deuterio , Óxido de Deuterio/metabolismo , Estabilidad de Enzimas , Humanos , Ligandos , Proteínas Mutantes/química , Estructura Secundaria de Proteína , Proteínas Recombinantes , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm(2)/s and 31 µm(2)/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.
Asunto(s)
Proteínas Bacterianas/química , Simulación de Dinámica Molecular , Receptores de Superficie Celular/química , Proteínas Bacterianas/metabolismo , Hidrodinámica , Concentración de Iones de Hidrógeno , Conformación Proteica , Receptores de Superficie Celular/metabolismo , Espectrometría de Fluorescencia , Thermus thermophilus , Trisacáridos/metabolismoRESUMEN
12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme-substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages.